Modification of gap junctional intercellular communication by changes in extracellular pH in Syrian hamster embryo cells

Studies were conducted to determine the effect of culture medium pH on gap junctional intercellular communication (GJIC) in early passage Syrian hamster embryo (SHE) cells. Previous studies have demonstrated that SHE cells cultured at a clonal density at pH 6.70 are morphologically transformed by carcinogens at a significantly higher frequency than cells cultured in media of higher pH. Several other cell characteristics consistent with promotion-like effects are observed with pH 6.70 culture of SHE cells. It was postulated that the promotion-like effects observed in SHE cells cultured at acidic pH are mediated in part by a reduction of GJIC. In this study, we evaluated GJIC in SHE cells by fluorescent dye coupling. Results from this study indicate that GJIC decreased as a function of decreased extracellular pH. Cells cultured at pH 6.70, 7.15 or 7.35 exhibited 47, 75 and 85% coupled cells respectively. The decrease in dye coupling occurred by 24 h after switching the cells from pH 7.15 to 6.70 medium. The decreased GJIC observed at pH 6.70 was not due to changes in cell proliferation and was reversible within 24 h when pH 6.70 cultures were refed with pH 7.15 medium. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate inhibited SHE cell GJIC in a pH-dependent manner with cells at pH 6.70 exhibiting the greatest inhibition by TPA and cells at pH 7.35 being unresponsive. The effect of pH on GJIC in SHE cells is consistent with the pH-dependent response to chemically induced morphological transformation and may be mechanistically related to this phenomenon.     

Influence of pH on the modification of thiols by carbamoylating agents and effects on glutathione levels in normal and neoplastic cells

Summary In previous studies, we have suggested that the selective inhibitory effect of sodium cyanate (NaOCN) on hepatoma metabolism may be due to the lower pH observed in tumors relative to normal tissues. Lower pH might enhance the action of NaOCN by increasing the formation of isocyanic acid and carbamoylation of sulfhydryl groups. In the present work, studies were conducted on the effect of pH on the carbamoylation of sulfhydryl groups. The data indicated that carbamoylation of the sulfhydryl group of glutathione by NaOCN was enhanced by decreasing the pH from 7.4 to 6.6. A less pH-dependent response was observed with organic isocyanates. However, all reactions were reversible after the pH was increased by the addition of base. Kinetic studies showed that the rate of the reaction is very rapid, a maximal effect occurring within the first 10 min. Dose-dependent modifications of cellular glutathione by NaOCN and organic isocyanates were observed in human HT29 colon tumor cells, rat HTC hepatoma cells, and rat hepatocytes. The rate of carbamoylation of the glutathione sulfhydryl group in cells was similar to that of pure glutathione (GSH). The effect of buthionine sulfoximine on GSH levels in cells was at least as great as that of sodium cyanate, but only the latter showed inhibitory effects on macromolecular synthesis; these were very rapid, pH-dependent, and reversible in tumor cells. Our results suggest that cellular sulfhydryl group(s) other than that of GSH might be involved in the effect of NaOCN on macromolecular synthesis.     

Cytotoxic effect of RB 6145 in human tumour cell lines: dependence on hypoxia, extra- and intracellular pH and drug uptake.

Low pH and hypoxia are a common feature of many solid tumours. This study examined the effect of these two conditions on the cytotoxic properties of the bifunctional agent RB 6145, the prodrug of RSU 1069. The effect of acidic pH on RB 6145 toxicity was examined in six human tumour cell lines under hypoxic conditions and was found to have little effect in HT 29, A549, U373 and HT 144 cells. Treatment was for 1 h at 37 degrees C, pH 6.4 or 7.4. Significant potentiation of RB 6145 toxicity was observed in SiHa cells (enhancement ratio; ERpH approximately 1.6) and in U1 cells (ERpH approximately 1.4). In these two cell lines the potentiation of RB 6145 toxicity arising from hypoxia was large, with ERHyp approximately 11 and 15 in SiHa and U1 cells respectively. SiHa cells, which show a pH effect and HT 29 cells, which do not, were chosen for further comparative studies of drug uptake )nd regulation of intracellular pH. High-performance liquid chromatography (HPLC) determinations of the uptake of RB 6145 and its dervatives showed that in SiHa cells, intracellular to extracellular drug concentration ratio (Ci/Ce) at 1 h was approximately 40% higher at pH 6.4 than at pH 7.4, whereas in HT 29 cells Ci/Ce was approximately 25% lower. Under conditions of acidic extracellular pH, regulation of pH was somewhat less effective in SiHa cells, where pHi dropped to within 0.2 pH units of the extracellular pH over a 2.5 h treatment at pH 6.4. It seems likely that increased drug uptake was at least part of the basis for the observed potentiation of RB 6145 toxicity in SiHa cells. A model which would better explain the results for both cell lines might also include the possibility that low pH per se potentiates cytotoxic damage to a modest extent and that it is offset or augmented by altered uptake in HT 29 and SiHa cells respectively.     

A differential low pH effect on tumour cells grown in vivo and in vitro when treated with hyperthermia

The effectiveness of low extracellular pH in sensitizing cells to heat was studied using SCK mammary carcinoma cells of A/J mice. Solid tumours of 400-600 mg or cells grown in vitro for less than 13 weeks were dispersed to single cells and the in vivo- or in vitro-derived cells were suspended in a medium of pH 7.2 or 5.5-6.6 and then heated at 41-44 degrees C in vitro using a water bath. The dispersion procedure did not alter the heat sensitivity of these cells, but acidic medium of pH below 6.6 caused an increase in heat sensitivity. This effect of low pH decreased with increasing temperature of heating for both cell types. However, the effect was much smaller on in vivo-derived cells than that on in vitro-derived cells for any heating temperature tested. This reduction of the pH effect was also observed for cells derived from larger tumours as well as tumours at an early stage of growth in which the internal milieu was not acidic. This indicates that cellular adaptation to low intratumour pH was not the cause of the reduced pH effect; instead, factors other than low pH must cause the reduced pH effect seen in tumour-derived cells.     

Carcinogen binding to various types of dietary fiber

The percent of the carcinogen 1,2-dimethylhydrazine (DMH) bound to a variety of fibers, such as wheat bran, corn bran, citrus pulp, citrus pectin, and alfalfa, was examined at pH values ranging from 1 to 12. The percent of DMH bound to wheat bran increased from 4% at PH 1 to 55% at pH 2 to 77% at pH 12. A sharp rise in carcinogen binding to corn bran occurred between pH 5% of the DMH was bound and pH 8 where 51% of the DMH was bound. The percent of DMH bound to dehydrated citrus pulp also increased as the pH increased with 10% binding observed at pH 1 and with 57% binding observed at pH 12. Between pH 2 and pH 7, the percent of DMH bound to pectin decreased from 60 to 11%. As the pH became more basic, the percent of DMH bound to pectin increased to 42% at pH 12. The sharpest rise in the percent of DMH bound to alfalfa meal occurred between pH 10.5 and pH 12.0. Results from this experiment showed that the affinity to various types of dietary fibers for the colon carcinogen DMH was differentially affected by pH. These results suggested that the protective effect of certain types of dietary fiber against chemically induced colon cancer my in part be attributed to enhanced carcinogen binding by dietary fiber in the colon.     

Influence of low pH on the development and decay of 42°C thermotolerance in cho cells

The development, magnitude and decay of thermotolerance in Chinese hamster ovary cells heated to 42°C at pH 6.7 was examined. The cells were exposed to single or fractionated heat treatments with 0 to 168 hours elapsing between the treatments. Administration of a specified heat treatment in two fractions at low pH substantially reduced the lethal effect of heat. The rate of hyperthermic cell killing was reduced by a factor of approximately 2.5 in preheated cells compared to cells not receiving prior heat treatment. Tolerance to second heat treatments was apparent when 3 hours at 37°C separated the treatments, and was near maximum when 6 hours separated the treatments. Beginning about 96 hours after the initial heat treatment, resistance to second heat treatments began to decline, and was not evident when 168 hours at 37°C separated the treatments. In contrast to these results, a sparing effect as a result of dose fraction was not observed at pH 7.4. At pH 7.4, cells developed thermotolerance during the initial heat treatment and additional culturing at 37°C was without additional effect. Nevertheless, the sensitivity of cells to hyperthermia was greater at pH 6.7 than at pH 7.4. This pH sensitizing effect was more pronounced in cells exposed to single heat treatments than in cells exposed to fractionated treatments at 42°C.     

Influence of microenvironmental pH on adriamycin resistance

Resistance to Adriamycin (ADR) is frequently dependent upon enhanced efflux associated with the expression of the MDR1-encoded P membrane glycoprotein. Since enhanced expression of the MDR1 gene in ADR-resistant cells may be the result of spontaneous genetic mutation or amplification, it is presumed to be relatively stable and unalterable. Yet, reducing ADR efflux could increase sensitivity, and has been attempted using calcium channel blockers and other drugs. However, since the tumor cell microenvironment varies with respect to pH because of differences in vascularization, oxygenation, and metabolite clearance, the possibility exists that these factors could influence drug transport and the critical biochemical pathways which determine cytotoxicity, even in resistant cells. Using flow cytometric analysis of ADR fluorescence, the influx and efflux of 10 microM ADR dissolved in MES buffer (pH 6.5) and 4-(2-hydroxyethylene)-1-piperazineethanesulfonic acid buffer (pH 7.5 and 8.5) was measured in sensitive P388 and resistant P388/R84 cells in vitro. Substantially enhanced uptake of ADR was detected at alkaline pH in both cell populations, while the proportion of ADR-positive cells and the level of ADR uptake was decreased at lower pH. Acidification reduced ADR efflux, whereas alkalinization increased efflux when the uptake pH was 6.5 or 7.5. At uptake pH 8.5, the pH of the external buffer had little effect, even in resistant cells. In resistant cells in an alkaline microenvironment, ADR transport and retention were superior to that observed in sensitive cells in an acidic microenvironment. No differences were observed in ADR transport when the transmembrane pH gradient was equilibrated. These observations are especially relevant to the effect of ADR on tumor cell subpopulations that are acidic, and in which drug diffusion is inefficient. Efforts to alkalinize tumor cells prior to ADR therapy might reduce ADR resistance, even of genetic origin.     

Combined effect of pH and sodium cyanate on the inhibition of tumor cell proliferation and metabolism by BCNU and hyperthermia

Summary In previous studies, we have found that combined treatment with BCNU and sodium cyanate could have a greater effect on the survival of mice bearing B16 melanoma than treatment with either agent alone. With rat hepatoma and human colon cancer cells in culture, we have obtained evidence that the inhibition of cell proliferation by sodium cyanate is greater at pH 6.6 than at pH 7.4. In the present work, the effects of combination treatments on the proliferation of cancer cells were studied with cyanate, pH, BCNU, and hyperthermia. With HT29 human colon cancer cells, the inhibitory effect of BCNU (50–100 μg/ml) was greater when the cells were treated at pH 6.6 than at pH 7.4. The influence of pH appeared to be absent or minimal at lower or higher concentrations of BCNU. We confirmed our previous observation that the inhibition of proliferation of LS174T human colon cancer cells is greater at pH 6.6 than at pH 7.4, and we observed an inhibitory effect of BCNU (50 or 200 μg/ml). However, no more than additive effects were seen with combination treatment. An inhibitory effect of hyperthermia was seen for the incorporation of [³H]-leucine into protein of rat hepatoma cells (HTC) and for that of [³H]-thymidine into DNA of human colon cancer (HT29) cells. In neither case was the effect of hyperthermia significantly enhanced by treatment with sodium cyanate beyond that seen with one of the treatments alone. The data confirmed that the inhibitory effect of sodium cyanate on cell proliferation can be enhanced by a low pH but did not provide evidence for synergistic effects in combination with BCNU or hyperthermia.     

pH effects on the lifespan and transformation frequency of Syrian hamster embryo (SHE) cells

We have investigated the effects of modification of the growthmedia pH on Syrian hamster embryo (SHE) cellular lifespan andtransformation. Culturing SHE cells in Dulbecco's modified Eagle'smedium (DMEM) at pH 6.7 relative to pH 7.3 results in a 4-foldincrease in the lifespan of SHE cells before senescence occurs.This effect was observed both with the wild type SHE cell populationand with the morphological transformation sensitive subpopulationsderived from the wild type SHE cell mixture (c19 and cl17).The increase in SHE cell lifespan at pH 6.7 was observed bothas an increase in days in culture and an increase in the numberof total population doublings before cellular senescence occurred.The tumor promoting agent TPA increased the lifespan of theSHE cell population and one of the MT sensitive subpopulationswhen these cells were cultured in pH 7.3 medium. Interestingly,treatment of the SHE cell population, and the two MT sensitiveSHE cell clones with TPA at culture medium pH of 6.7 reducedthe lifespan of each cell type. Finally, culturing SHE cellsin medium of pH 6.7 increased the potential of the cells toundergo morphological transformation in response to treatmentwith carcinogens, demonstrating a potential connection betweenSHE cell lifespan and carcinogen induced morphological transformation,probably through a mechanism involving a pH induced block incellular differentiation. We believe this is the first reportdemonstrating the role of pH in controlling cellular lifespan.     

Variation of pH of mammary tumour cytosols: potential influence on steroid receptor assay

Mammary tumour cytosols prepared for steroid receptor assay showed pH values above or below 7.3-7.5 in 50% of the samples. An increase of 0.6 pH units was observed after the addition of dextran coated charcoal. To test the sensitivity of the assay systems to pH changes, breast cancer cytosols were adjusted to pH between 6.0-9.0 before incubation with ligand at 0 degrees C. ER was then separated by isoelectric focusing in a pH gradient between 9.5-3.5 in a routine assay including a dextran coated charcoal step before focusing. PgR was assayed by electrofocusing as well as by a conventional DCC method and Scatchard plot. Unadjusted control cytosols were run in parallel. At pH above or below 7.0-8.0 recovery of receptor was reduced, the loss being most prominent at low pH. At low pH levels Scatchard plots appeared to be of curvo-linear shape. The effect of low intracellular tumour pH on receptor-ligand affinity in vitro may be of great importance as well in vivo, and should be taken into consideration in the therapeutic situation.     

The effect of hypoxia and low pH on the cytotoxicity of chlorambucil.

Studies with mouse tumors have shown that the effectiveness of certain chemotherapeutic agents can be enhanced if they are used in appropriate combination with an anti-hypertensive drug such as hydralazine. This results in reduced tumor blood flow with, among other things, a consequent decrease in oxygenation and increase in acidity in the tumor tissue. The purpose of the present work was to determine to what extent hypoxia and low pH are involved in the mechanism of this effect for chlorambucil. V79-WNRE cells were exposed to various drug concentrations under aerobic or hypoxic conditions, pH 6.4 or 7.4. Measurements of cell survival following 1 hr exposure at 37 degrees C showed that pH 6.4 produced a large potentiation of cell killing by chlorambucil (ER = 4 approx.); hypoxia, on the other hand, had little effect. The potentiation was shown to be greatest for pH values below 7.0. HPLC measurements of drug uptake were made since it was anticipated that chlorambucil, a weak acid, might tend to accumulate in cells under conditions of low extracellular pH. It was found that at an extracellular pH of 6.4 the ratio of the intracellular (Ci) and extracellular (Ce) drug concentrations was increased 4.5 and 3.6 fold for aerobic and hypoxic conditions, respectively. This probably explains most, if not all, of the cell killing potentiation observed at low pH.     

Enhanced mutagenic activities of N-nitroso compounds in weakly acidic media

Mutagenesis induced by a number of N-nitrosamines in a liquidphase assay was enhanced 2–8 fold on going from a pH of7.4 to 6.5, with the degree of enhancement dependent on thecompound and the reaction conditions. In general, enhancementwas greatest at lower concentrations of mutagen and with compoundsof lower molecular weights. With dimmethylnitrosamine (DMN)part of this effect was due to an enhanced rate of metabolismat low concentrations of DMN at a pH of 6.5. However, even athigher concentrations of DMN, mutagenesis was more effectiveat the lower pH despite the fact that metabolism of DMN wastwice as rapid at a pH of 7.4 than 6.5. Directly acting mutagenssuch as N-nitroso-N-methylurea and N-methyl-N' -nitro-N-nitrosoguanidinewere 2–10 times more potent at pH values of between 5.5and 7.0 than at 7.4. No enhancement in the mutagenic activitiesof methyl methanesulfonate, benzo(a)pyrene or 3-methyl-cholanthrenewas observed on going from a pH of 7.4 to 6.5.     

Intracellular acidification is associated with enhanced morphological transformation in Syrian hamster embryo cells.

A series of studies has indicated that the frequency of morphological transformation induced by chemical carcinogens in early passage Syrian hamster embryo (SHE) cells is significantly higher when these cells are cultured in medium of reduced bicarbonate concentration and pH (6.70) compared with cells cultured in medium of higher pH. It has also been shown that intercellular gap junctional communication is decreased in these cells when they are cultured at pH 6.70 compared with medium of higher pH. The purpose of the studies reported here was to characterize the effect of changing extracellular pH on intracellular pH in SHE cells. The frequency of morphological transformation induced by benzo(a)pyrene was established at various extracellular pHs and compared with intracellular pH values. Cells cultured in medium of pH ranging from 6.70 to 7.35 were loaded with the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, and either the steady-state intracellular pH values or the kinetics of change in intracellular pH following refeeding of the cultures with medium of pH ranging from pH 6.70 to pH 7.35 was monitored via image analysis techniques. Results from these studies indicate that, at culture medium pH above 6.95, SHE cells were relatively insensitive to changes in extracellular pH, maintaining an intracellular pH of 7.30 to 7.35 in medium containing 0% serum or pH 7.05 to 7.10 in medium containing 20% fetal bovine serum. At extracellular pHs below 6.95, intracellular pH decreased and, in the presence of serum, equilibrated with extracellular pH. The decrease in intracellular pH was closely associated with an increase in benzo(a)pyrene-induced morphological transformation frequency observed in parallel studies. These results indicate that SHE cells have active intracellular pH regulatory activities and suggest that intracellular acidification plays a role in the increased frequency of transformation observed in SHE cells cultured under acidic conditions.     

靶动脉灌注NaHCO3提高部分抗肿瘤药物疗效的基础及临床研究

Objective: To observe the influence of pH value on the proliferation of LAK cells and on the killing effect of rIL-2,IFN-α2b, TNF-α, LAK cells and doxorubicin on malignant tumor cells, and investigate the possibility of increasing the efficacy of rIL-2 or IFN-α2b and doxorubicin by infusing sodium bicarbonate (NaHCO3) through target arteries. Methods: Separating single nucleus cells from peripheral blood of healthy men, and observing the influence of pH on the activation of single nucleus cells by rIL-2. MTT assay was used to measure the killing effect of rIL-2, IFN-α2b and TNF-α on 7404 cells and the increased effect of doxorubicin on rIL-2 and IFN-α2b, the cytotoxity of LAK cells in different pH. Forty-two patients with advanced primary liver cancer were obtained by stratified random, NaHCO3, rIL-2/IFN-α2b and doxorubicin were infused through target arteries. The efficacy was estimated after two cycles. Results: The conditions of pH 7.3 and pH 7.6 in vitro helped the proliferation of LAK cells and the killing effect of rIL-2, IFN-α2b and LAK cells on 7404 cells. In the condition of pH 6.8 there was almost no killing effect for LAK cells. In the condition of pH 7.0, 7.2, 7.4 and 7.6, the killing rate of TNF-α to 7404 cells increased by degrees, and in pH 7.4 the killing effect was the optimum. After two cycles treatments in the 42 patients with advanced primary liver cancer,the response rate (CR PR) was 88% (37/42). The median overall response and median overall survival were increased, and no complication associated with infusing sodium bicarbonate was observed. Conclusion: The killing effect of rIL-2, IFN-α2b, TNF-αand doxorubicin on malignant tumor cells was enhanced by increasing the pH value.     

Distribution of endocytosed molecules to intracellular acidic environments correlates with immunotoxin activity

We have investigated the internalization to low pH intracellular compartments of transferrin (Tfn), diphtheria toxin (DT) and of anti-cell surface antibodies (MAb) by a cytofluorometric assay based on low pH quenching of fluorescein (FITC) emission. FITC-labelled Tfn, anti-CD3, anti-CD5 and anti-Thy 1.2 MAb internalization resulted in a progressively lower FITC quenching effect. Following internalization, a distinction could be made between molecules that enter low pH compartments without undergoing intracellular degradation (e.g., Tfn, anti-CD3 MAb) and molecules that are internalized through low pH organelles and are then degraded within the cell (e.g., DT). A strict correlation was observed between quenching of internalized FITC-protein fluorescent emission and the cytotoxic activity of DT-based immunotoxins (IT).     

The sensitivity of a malignant cell line to hyperthermia (42 degrees C) at low intracellular pH.

The postulate that low intracellular pH acts as a preconditioner for the destructuve effects of hyperthermia (42 degrees C) was examined, using a heat-sensitive line of malignant cells derived from rat mammary gland (SDB). Intracellular pH (pHi) was measured indirectly, from the distribution of the weak, non-metabolizable organic acid 5,5-dimethyl-2,4-oxazolidinedione (DMO) between intra- and extra-cellular water. Respiration, aerobic and anaerobic and anaerobic glycolysis of the cells were studied at normal pHi (pH 7-0-7-4) or at low pHi (pH 6-2-6-6) and at 38 degrees C or 42 degrees C over 6 h in Warburg manometers; the ability of the cells to replicate in culture was examined after 3 h or 6 h incubation in the flasks. The relationship between pHi and extracellular pH (pHe) depended upon the buffer system used and the exact pH in question; no assumption regarding pHi based only on pHe measurement could be made. At 38 degrees C and low pHi, the Pasteur effect became negative due to a relatively greater inhibition of anaerobic than aerobic glycolysis. Respiration was unaffected and cell replicative ability unimpaired. At 42 degrees C and normal pHi, respiration was totally inhibited after 4 h and the Pasteur effect was decreased, in this case due to a compensatory increase in aerobic glycolysis without alteration in anaerobic CO2 production. Low pHi in the presence of hyperthermia enabled cell respiration to continue at a reduced level with no further change in glycolysis. There was delayed cell replication after 3 h at 42 degrees C and inability to multiply following 6 h hyperthermia: low pHi did not influence these results. It is concluded that with these cancer cells, pHi values maintained in the region of 1-0 pH unit below normal for 6 h had no deleterious effect on the cells. No sensitizing effect of the low pHi for the destructive effect of hyperthermia on the cells was observed.     

Continuous noninvasive monitoring of pH and temperature in rat Walker 256 carcinoma during normoglycemia and hyperglycemia

The extracellular pH and temperature of Walker 256 carcinoma and of normal subcutaneous tissue were measured continuously in unanesthetized female Sprague-Dawley rats for up to 20 hours following glucose or galactose administration. The pH was monitored with flexible glass electrodes contained in micropore chambers implanted in the flank of a rat. Temperature was measured with miniature thermistor probes incorporated in the tumor or in subcutaneous tissue. The pH in the untreated Walker 256 carcinoma decreased linearly from approximately 7.3 to 6.2 with increasing tumor mass up to 50 g. Administration of glucose (6 g/kg body wt, ip) in tumor-bearing rats increased glucose concentrations in blood and tumor, as well as lactic acid concentration in tumor, and had no significant effect on lactic acid concentration in blood. Plasma volume was not affected by either glucose or galactose loading as compared to that in rats given saline alone. However, the blood viscosity increased by up to 30% within 30 minutes after galactose injection, but not after glucose injection, and this significant difference in viscosities persisted for approximately 6 hours after glucose and galactose injections. In small tumors (less than 10 g), a decrease of up to 1 pH unit was observed within 6 hours after glucose administration, and the return of pH to pretreatment values began about 10 hours after glucose injection. Response of large ulcerated tumors (greater than 20 g) was not as uniform; the pH decreased by about 0.5 to 1 pH unit for only a brief period. After galactose injection, pH in some tumors remained unchanged, whereas in others an average decrease of about 0.2 pH units was observed. The pH in normal tissue was not affected by glucose or galactose administration. Both glucose and galactose decreased tumor temperature by about 7 degrees C.     

Na+/H+ exchanger activity is increased in doxorubicin-resistant human colon cancer cells and its modulation modifies the sensitivity of the cells to doxorubicin

Multidrug resistant (MDR) tumor cells exhibit an altered pH gradient across different cell compartments, which favors a reduced intracellular accumulation of antineoplastic drugs and a decreased therapeutic effect. In our study, we have observed that the activity and expression of Na+/H+ exchanger (NHE), which is involved in the homeostasis of intracellular pH (pHi), are increased in doxorubicin-resistant (HT29-dx) human colon carcinoma cells in comparison with doxorubicin-sensitive HT29 cells. The pH(i) was significantly higher in HT29-dx cells, which accumulated less doxorubicin than HT29 cells. The NHE inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA) significantly reduced the pHi value and increased the intracellular accumulation of doxorubicin in both cell populations: in the presence of EIPA HT29-dx cells accumulated as much drug as control HT29 cells. On the other hand, monensin, a Na+/H+ ionophore mimicking NHE activation, and phorbol 12-myristate 13-acetate (PMA), which stimulates NHE, significantly increased the pHi and decreased the drug accumulation in HT29 cells to values similar to those observed in control HT29-dx cells. EIPA potentiated the cytotoxic effect of doxorubicin in HT29 cells, and made HT29-dx cells as sensitive to the cytotoxic effect of the drug as control HT29 cells. Instead, PMA and monensin made HT29 cells as insensitive to doxorubicin as HT29-dx cells. These results suggest that in MDR cells the higher cytosolic pH is likely to decrease drug accumulation, and that such resistance can be reverted by inhibiting the NHE activity. This result opens the possibility to revert MDR with the clinical use of NHE inhibitors.     

Increased therapeutic efficacy of intra-arterial carboquone chemotherapy on a limb tumor in rats, by using an acidic vehicle adjusted with lactate

We previously reported that the cytotoxicity of carboquone (CQ) was potentiated in vitro and in vivo under acidic conditions. In this study, an acidic vehicle adjusted with lactate at various low pHs was used for CQ intra-arterial (i.a.) injection, in order to enhance the antitumor effects of i.a. CQ chemotherapy. Treatments were evaluated in Wistar/KA rats bearing a limb tumor 5 days after the inoculation of 3 x 10(6) syngeneic RBT-1 tumor cells into the hind limb. In chemotherapy experiments using an intrafemoral injection of CQ at 1.5 mg/kg in phosphate-buffered saline (PBS, pH 7.4) or in an acidic vehicle at pH 5.0 or 6.0, the antitumor effects seen in rats given CQ in acidic vehicles, evaluated by tumor weight 14 days after treatment, were significantly greater than that seen in rats given CQ in PBS. There were no significant differences either in changes of body weight or in the number of leukocytes after treatment between the groups given CQ in PBS and in an acidic vehicle at pH 6.0. Although in the group given CQ at 2.0 mg/kg in PBS, the antitumor effect was the same as that observed in rats given CQ at 1.5 mg/kg in an acidic vehicle at pH 6.0, the side effects observed in the former group were much severer than in the latter group. These data suggest that the antitumor effect of i.a. CQ chemotherapy can be potentiated by using an acidic vehicle.     

Determination of extracellular pH and tissue temperature in transplantable rat tumors by use of inductive loop telemetry

Radiotelemetry was used to determine changes in extracellular pH and tissue temperature during tumor development in rats of an inbred WAB substrain. A progressive decrease in the pH of the tumor tissue compared with the pH level in control tissue was observed, so that by 4 days after tumor implantation the tumor tissue was about 0.4 pH units lower than control values. Thereafter, tumor pH rose progressively to reach a value comparable with control levels at 8 days, when the experiment was terminated. No significant difference was detected between the temperature in the tumor tissue and the temperature in the control tissue. Therefore, the observed changes in pH appeared to be independent of temperature. Studies on the rate of tumor cell proliferation during the experiments demonstrated a significant inverse relationship between tumor cell mitotic rate and pH, with the highest rates of cell proliferation occurring at the time of lowest pH values.